Ubiquitin (Ub), a highly conserved 76-residue protein, is present in eukaryotic cells either free or covalently joined to a great variety of proteins. The posttranslational coupling of ubiquitin to other proteins is catalyzed by a family of Ub-conjugating (E2) enzymes and involves formation of an isopeptide bond between the C-terminal Gly residue of ubiquitin and the .epsilon.-amino group of a Lys residue in an acceptor protein. One function of ubiquitin is to mark proteins destined for selective degradation. Ubiquitin was also shown to have a chaperone function, in that its transient (cotranslational) covalent association with specific ribosomal proteins promotes the assembly of ribosomal subunits.
Unlike branched Ub-protein conjugates, which are formed posttranslationally, linear Ub-protein adducts are formed as the translational products of natural or engineered gene fusions. Thus, in the yeast Saccharomyces cerevisiae for example, ubiquitin is generated exclusively by proteolytic processing of precursors in which ubiquitin is joined either to itself, as in the linear polyubiquitin protein Ubi4, or to unrelated amino acid sequences, as in the hybrid proteins Ubi1-Ubi3. In growing yeast cells, ubiquitin is generated largely from the Ubi1-Ubi3 precursors whose "tails" are specific ribosomal proteins. The polyubiquitin (UBI4) gene is dispensable in growing cells but becomes essential (as the main supplier of ubiquitin) during stress. The lack of genes encoding mature ubiquitin, and the fusion structure of ubiquitin precursors in yeast are characteristic of other eukaryotes as well.
Ub-specific, ATP-independent proteases capable of cleaving ubiquitin from its linear or branched conjugates have been detected in all eukaryotes examined but not in bacteria such as Escherichia coli, which lack ubiquitin and Ub-specific enzymes. Miller et al. (Biotechnology 1: 698-704 (1989)) have cloned a S. cerevisiae gene, named YUH1, encoding a Ub-specific protease that cleaves ubiquitin from its relatively short C-terminal extensions but is virtually inactive with larger fusions such as Ub-.beta.-galactosidase (Ub-.beta.gal). Wilkinson et al. (Science 246: 670-673 (1989)) have also cloned a cDNA encoding a mammalian homolog of the yeast Yuh1 protease. Tobias and Varshavsky (J. Biol. Chem. 266: 12021-12028 (1991)) reported the cloning and functional analysis of another yeast gene, named UBP1, which encodes a Ub-specific processing protease whose amino acid sequence is dissimilar to those of the Yuh1 protease and other known proteins. Unlike YUH1 and its known homologues in other species, Ubp1 deubiquitinates ubiquitin fusion proteins irrespective of their size or the presence of an N-terminal ubiquitin extension.